Listeria monocytogenes is a gram‐positive facultative intracellular bacterium, which replicates in the cytoplasm of myeloid cells. Interferon β (IFNβ) has been reported to play an important role in the mechanisms underlying Listeria disease. Although studies in murine cells have proposed the bacteria‐derived cyclic‐di‐AMP to be the key bacterial immunostimulatory molecule, the mechanism for IFNβ expression during L. monocytogenes infection in human myeloid cells remains unknown. Here we report that in human macrophages, Listeria DNA rather than cyclic‐di‐AMP is stimulating the IFN response via a pathway dependent on the DNA sensors IFI16 and cGAS as well as the signalling adaptor molecule STING. Thus, Listeria DNA is a major trigger of IFNβ expression in human myeloid cells and is sensed to activate a pathway dependent on IFI16, cGAS and STING. Synopsis The innate immune system detects microbes and induces protective and pathological responses. Here, it is shown that bacterial DNA is the molecule‐triggering expression of interferon β during infection with Listeria monocytogenes in human myeloid cells and that this is dependent on the DNA sensors IFI16 and cGAS. Bacterial DNA is the molecule stimulating IFNβ expression during L. monocytogenes infection in human cells Listeria‐induced IFNβ expression correlates with bacteriolysis in the macrophage cytosol, and bacterial DNA associates with IFI16 and cGAS Expression of IFNβ in Listeria‐infected cells proceeds through a pathway dependent on IFI16, cGAS and STING While bacterium‐derived cyclic‐di‐AMP triggers interferon responses in murine cells, Listeria monocytogenes‐infected human myeloid cells utilize an innate immune sensory pathway that directly recognizes bacterial DNA.